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Abstract:

The present invention provides antimicrobial solutions that comprise at
least one alcohol, at least one antimicrobial agent and at least one
chelator and/or anticoagulant. Also provided are methods for rapidly
reducing a microbe or a virus from surfaces including surfaces of
indwelling medical devices and organic surfaces such as skin and sutures,
and inorganic surfaces such as hospital equipment, pipelines etc.

Claims:

1.-40. (canceled)

41. A device, wherein a surface of the device has been treated to render
the surface more resistant to growth of microbial organisms, the
treatment comprising the steps of contacting the surface an antimicrobial
solution comprising ethanol, an EDTA and either minocycline or
trimethoprim, wherein the concentration of ethanol is in the range of 10%
to 45% (v/v), the ethanol, EDTA and minocycline or trimethoprim being
present in amounts effective to eradicate Candida parapsilosis or
methicillin resistant S. aureas embedded in a biofilm.

42. The device of claim 41, wherein the concentration of the ethanol is
in the range of 10-40%.

43. The device of claim 42, wherein the concentration of ethanol is in
the range of 15-30%.

44. The device of claim 43, wherein the concentration of the ethanol is
about 25%.

54. The device of claim 41, further defined as the surface of a pipe or
pipeline, a floor, a table-top, a counter-top, hospital equipment, or a
wheel chair, an oil pipeline, a water pipeline, an ice machine pipe, or a
beverage dispensing pipe.

Description:

[0001] The present application claims the benefit of the filing date of
U.S. Provisional Patent Application Ser. No. 60/476,555, filed on Jun. 6,
2003. The entire text of the above-referenced disclosure is specifically
incorporated herein by reference without disclaimer.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The present invention relates generally to the fields of medicine
and microbiology. More particularly, it concerns methods of reducing
microbial organisms from indwelling medical devices, medical equipment
and other surfaces.

[0004] 2. Description of Related Art

[0005] Medical devices, such as vascular catheters, have improved the
quality of medical care. However, infections resulting from the
colonization of organisms embedded in biofilm are the most frequent
complication associated with the use of these and other indwelling and/or
prosthetic devices. In fact, infections are the most serious
complications associated with indwelling central venous catheters (CVCs)
(Maki et al., 1998). It is estimated that more than 200,000
catheter-related bloodstream infections (CRBSI) occur annually in the
United States alone (Kluger et al., 1999). Staphylococcus epidermidis,
Staphylococcus aureus and Candida species are the leading organisms
causing CRBSI (Maki et al., 1998; Raad et al., 2002).

[0006] Because intralumenal colonization is the major source for the
migration of organisms leading to bloodstream infections in long-term
silicone catheters (Raad et al., 1993), recent guidelines by the CDC and
Infectious Diseases Society of America have proposed the use of
intralumenal antimicrobial lock solutions for the prevention and
treatment of CRBSI (Mermel et al., 2001; Centers for Disease Control and
Prevention, 2002). Most long-term CVCs are typically flushed with
heparin. An antimicrobial/anticoagulant combination consisting of
vancomycin/heparin with and without ciprofloxacin was shown to reduce the
risk of catheter-related bacteremia caused by gram-positive organisms
(Carratala et al., 1999; Henrickson et al., 2002; Schwartz et al., 1990).
However, with the rise of incidences of infection by vancomycin resistant
gram-positive bacteria, concerns have been raised over the use of
vancomycin flush solutions and their potential for increasing the risk of
vancomycin resistance (Spafford et al., 1994).

[0007] Recently the present inventor demonstrated that a flush solution
comprising minocycline and EDTA (M-EDTA) is highly efficacious in
preventing catheter-related colonization, bacteremia and endocarditis in
rabbits (Raad et al., 2002). When compared to a heparin flush solution,
M-EDTA was found to decrease the risk of catheter-related colonization
and infection in hemodialysis patients as well as pediatric cancer
patients (Bleyer et al., 2000; Chatzinikolaou et al., 2002). EDTA has an
equivalent anticoagulant activity to heparin (Reardon et al., 1991). An
anticoagulant in flush solutions is necessary to prevent the thrombotic
occlusion of the catheter lumen.

[0008] Although M-EDTA has been found to be efficacious in preventing
CRBSI, this solution may not be applicable given some of the limitations
of the real world of clinical practice. In the animal and clinical
studies, the M-EDTA lock solution was required to be exposed to the
surface of the indwelling medical device, such as the lumen of catheters,
for at least 4 hours. In vitro studies have also shown that M-EDTA
requires at least 4 hours of dwell time to eradicate organisms that
colonize the lumen of the catheter (see in particular data in U.S. Pat.
No. 5,362,754, columns 11 and 12, and Tables 3, 4 and 5 as well as in
U.S. Pat. No. 5,688,516, columns 15 and 16, and Tables 3, 4, and 5).
Providing a four hour exposure time to reduce microbes using the M-EDTA
solution is usually not possible in critically ill patients who require
continuous infusion therapy, including parenteral nutrition.

[0009] Thus, there is an acute need in the art to develop compositions and
methods for rapid reduction and/or eradication of microbes from
indwelling medical devices without interruption of the use of the device
in patients for too long a period. In addition, there is also a need for
better and improved antimicrobial compositions.

SUMMARY OF THE INVENTION

[0010] The present invention overcomes these and other limitations in the
art and provides compositions that reduce or eradicate microbial agents
from surfaces wherein the compositions comprise at least one
antimicrobial agent, at least one chelator and/or anticoagulant, and at
least one alcohol. The present invention also provides methods to rapidly
reduce or eradicate microbial agents from surfaces.

[0011] Therefore, provided are antimicrobial solutions comprising at least
one alcohol, at least one antimicrobial agent and at least one chelator
and/or anticoagulant. "Antimicrobial agents" that are comprised in the
solutions of the present invention include antibacterial agents,
antifungal agents, antiviral agents as well as antiseptic agents. These
components are present in effective amounts to reduce microbial growth.

[0012] In some embodiments of the invention, the antimicrobial agent is an
antibacterial agent. While any antibacterial agent may be used in the
preparation of the instant antimicrobial solutions, some non-limiting
exemplary antibacterial agent(s) include those classified as
aminoglycosides, beta lactams, quinolones or fluoroquinolones,
macrolides, sulfonamides, sulfamethaxozoles, tetracyclines,
streptogramins, oxazolidinones (such as linezolid), clindamycins,
lincomycins, rifamycins, glycopeptides, polymxins, lipo-peptide
antibiotics, as well as pharmacologically acceptable sodium salts,
pharmacologically acceptable calcium salts, pharmacologically acceptable
potassium salts, lipid formulations, derivatives and/or analogs of the
above.

[0013] Each of these classes of antibacterial agents have different
mechanisms of action and are represented by several antibiotics a
discussion of which is presented below. However, the skilled artisan will
recognize that the invention is in no way limited to the agents set forth
here and that these agents are described merely as examples.

[0014] The aminoglycosides are bactericidal antibiotics that bind to the
30S ribosome and inhibit bacterial protein synthesis. They are typically
active against aerobic gram-negative bacilli and staphylococci. Exemplary
aminoglycosides that may be used in some specific aspects of the
invention include amikacin, kanamycin, gentamicin, tobramycin, or
netilmicin.

[0015] Beta lactams are a class of antibacterials that inhibit bacterial
cell wall synthesis. A majority of the clinically useful beta-lactams
belong to either the penicillin group (penam) or cephalosporin (cephem)
groups. The beta-lactams also include the carbapenems (e.g., imipenem),
and monobactams (e.g., aztreonam). Inhibitors of beta-lactamase such as
clavulanic acid and its derivatives are also included in this category.

[0016] Non-limiting examples of the penicillin group of antibiotics that
may be used in the solutions of the present invention include
amoxicillin, ampicillin, benzathine penicillin G, carbenicillin,
cloxacillin, dicloxacillin, piperacillin, or ticarcillin, etc. Examples
of cephalosporins include ceftiofur, ceftiofur sodium, cefazolin,
cefaclor, ceftibuten, ceftizoxime, cefoperazone, cefuroxime, cefprozil,
ceftazidime, cefotaxime, cefadroxil, cephalexin, cefamandole, cefepime,
cefdinir, cefriaxone, cefixime, cefpodoximeproxetil, cephapirin,
cefoxitin, cefotetan etc. Other examples of beta lactams include mipenem
or meropenem which are extremely active parenteral antibiotics with a
spectrum against almost all gram-positive and gram-negative organisms,
both aerobic and anaerobic and to which Enterococci, B. fragilis, and P.
aeruginosa are particularly susceptible.

[0017] Examples of beta lactamase inhibitors include clavulanate,
sulbactam, or tazobactam. In some aspects of the present invention, the
antibacterial solutions may comprise a combination of at least one beta
lactam and at least one beta lactamase inhibitor.

[0018] Macrolide antibiotics are another class of bacteriostatic agents
that bind to the 50S subunit of ribosomes and inhibit bacterial protein
synthesis. These drugs are active against aerobic and anaerobic
gram-positive cocci, with the exception of enterococci, and against
gram-negative anaerobes. Exemplary macrolides include erythromycin,
azithromycin, clarithromycin.

[0020] Sulphonamides are synthetic bacteriostatic antibiotics with a wide
spectrum against most gram-positive and many gram-negative organisms.
These drugs inhibit multiplication of bacteria by acting as competitive
inhibitors of p-aminobenzoic acid in the folic acid metabolism cycle.
Examples include mafenide, sulfisoxazole, sulfamethoxazole, and
sulfadiazine.

[0021] The tetracycline group of antibiotics include tetracycline
derivatives such as tigecycline which is an investigational new drug
(IND), minocycline, doxycycline or demeclocycline and analogs such as
anhydrotetracycline, chlorotetracycline, or epioxytetracycline. The
present inventors have previously shown that minocycline has a higher
penetration of the microbial biofilm layer than vancomycin and that EDTA
is unique in effectively preventing and dissolving polysaccharide-rich
microbial glycocalyx (U.S. Pat. No. 5,362,754).

[0022] The streptogramin class of antibacterial agents is exemplified by
quinupristin, dalfopristin or the combination of two streptogramins.

[0023] Drugs of the rifamycin class typically inhibit DNA-dependent RNA
polymerase, leading to suppression of RNA synthesis and have a very broad
spectrum of activity against most gram-positive and gram-negative
bacteria including Pseudomonas aeruginosa and Mycobacterium species. An
exemplary rifamycin is rifampicin.

[0024] Other antibacterial drugs are glycopeptides such as vancomycin,
teicoplanin and derivatives thereof. Yet other antibacterial drugs are
the polymyxins which are exemplified by colistin.

[0025] In addition to these several other antibacterial agents such as
prestinomycin, chloramphenicol, trimethoprim, fusidic acid,
metronidazole, bacitracin, spectinomycin, nitrofurantion, daptomycin or
other leptopeptides, oritavancin, dalbavancin, ramoplamin, ketolide etc.
may be used in preparing the compositions described herein. Of these,
metronidazole is active only against protozoa, such as Giardia lamblia,
Entamoeba histolytica and Trichomonas vaginalis, and strictly anaerobic
bacteria. Spectinomycin, is a bacteriostatic antibiotic that binds to the
30S subunit of the ribosome, thus inhibiting bacterial protein synthesis
and nitrofurantoin is used orally for the treatment or prophylaxis of UTI
as it is active against Escherichia coli, Klebsiella-Enterobacter
species, staphylococci, and enterococci.

[0029] In some embodiments of the invention, the antiseptic agent is as
set forth in the specification of U.S. Provisional Application Ser. No.
60/261,447, U.S. Provisional Application Ser. No. 60/316,165, and U.S.
Non-Provisional Patent Application Ser. No. 10/044,842, incorporated
herein by reference in their entirety. Thus, in some embodiments the
antiseptic agent comprises a basic reagent and a dye.

[0030] The basic reagent may be a guanidium compound, a biguanide, a
bipyridine, a phenoxide antiseptic, an alkyl oxide, an aryl oxide, a
thiol, a halide, an aliphatic amine, or an aromatic amine. In some
specific aspects, the basic reagent is a guanidium compound. Non-limiting
examples of guanidium compounds include chlorhexidine, alexidine,
hexamidine. In other specific embodiments, the basic reagent is a
bipyridine. One example of a bipyridine is octenidine. In yet other
aspects, the basic reagent is a phenoxide antiseptic.

[0031] The dye may be a triarylmethane dye, a monoazo dye, a diazo dye, an
indigoid dye, a xanthene dye, an anthraquinone dye, a quinoline dye, an
FD&C dye. Non-limiting examples of triarylmethane dye include gentian
violet, crystal violet, ethyl violet, or brilliant green. Exemplary
monoazo dyes inlude FD&C Yellow No. 5, or FD&C Yellow No. 6. Other
non-limiting examples of FD&C dye include Blue No. 1 or Green No. 3. One
non-limiting example of diazo dyes is D&C Red No. 17. An example of an
indigoid dye is FD&C Blue No. 2. An examples of a xanthene dye is FD&C
Red No. 3; of an anthraquinone dye is D&C Green No. 6; and of an
quinoline dye is D&C Yellow No. 1.

[0032] Other examples of antiseptics that may be used to prepare the
antimicrobial solutions of the invention are the phenoxide antiseptics
such as clofoctol, chloroxylenol or triclosan. Still other antiseptic
agents that may be used to prepare the amntimicrobial solutions of the
invention are gendine, genlenol, genlosan, or genfoctol.

[0033] One of skill in the art will appreciate that one can use one or
more of the antimicrobial agents including one or more antibacterial
agent, and/or one or more antifungal agent, and/or one or more antiviral
agent, and/or one or more antiseptic agent, and/or combinations thereof.

[0035] Alternatively, one may use at least one anticoagulant such as
heparin, hirudin, EGTA, EDTA, urokinase, streptokinase, hydrogen peroxide
etc., in the preparation of the antimicrobial solutions of the invention.

[0036] A variety of alcohols are contemplated as useful in the preparation
of the instant antimicrobial solution, and include any antimicrobially
active alcohol. Non-limiting examples of alcohols include ethanol,
methanol, isopropanol, propylene glycol, benzyl alcohol, chlorobutanol,
phenylethyl alcohol, and the like. The concentration of the alcohol is
preferably in the range of 5%-80% (v/v), more preferably in the range of
10% to 50%, more preferably in the range of 15% to 40%, more preferably
in the range of 20% to 30%, with the most preferable being about 25%.
Thus, the more preferred concentration of alcohol will include 5%, 6%,
7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,
70%, 75%, or 80% (v/v) of the alcohol in the preparation of the instant
antimicrobial solutions. This includes the use of intermediate
concentrations of alcohol such as 11%, 22.5%, 26% and the like.

[0037] One of skill in the art will appreciate that the solutions of the
instant invention can comprise various combinations of at least one
alcohol, at least one antimicrobial agent, and at least one
chelator/anticoagulant. In some specific embodiments, the solution of the
invention comprises at least one alcohol, at least one tetracycline and
at least one chelator/anticoagulant. In a specific aspect, such an
antimicrobial solution comprises ethanol, at least one tetracycline and
EDTA or heparin.

[0038] In other specific aspects, such a solution comprises ethanol,
minocycline and EDTA or heparin. In one embodiment of this aspect, the
concentration of minocycline is 0.001 mg/ml to 100 mg/ml. In another
embodiment, the concentration of minocycline is about 3 mg/ml. In another
aspect, the concentration of EDTA is in the range of 10-100 mg/ml. In one
embodiment of this aspect, the concentration of EDTA is about 30 mg/ml.

[0039] The invention also provides methods for reducing microbial
organisms from a surface comprising: a) obtaining an antimicrobial
solution of the invention as set forth above; and b) contacting the
surface with the antimicrobial solution, whereby said contacting reduces
microbial organisms from the surface.

[0040] In one embodiment of the method, the contacting is performed for 4
hours or less. In other embodiments of the method, the contacting is
performed for 2 hours or less, for 1 hour or less, for 30 minutes or
less, or for 15 minutes or less.

[0041] In another aspect, the method further comprises eradicating
microbes from the surface wherein the contacting is performed for about
15 minutes or more.

[0042] The methods of the invention can be used to reduce microbial agents
from the surface of a medical device such as a catheter, an endotracheal
tube, a nephrostomy tube, a biliary stent, an orthopedic device, a
prosthetic valve, a medical implant, dental devices or dental implants,
cardiac assist devices, vascular grafts, tracheostomy, ventriclulostomy
devices, or intrathecal devices. In some aspects, the catheter is an
indwelling catheter such as a central venous catheter, a peripheral
intravenous catheter, an arterial catheter, a Swan-Ganz catheter, a
hemodialysis catheter, an urinary catheter, a peritoneal catheter, an
umbilical catheter, a percutaneous nontunneled silicone catheter, a
cuffed tunneled central venous catheter or a subcutaneous central venous
port.

[0043] In other embodiments, the methods of the invention are useful in
reducing microbial agents from a surface such as an organic surface or an
inorganic surface. An organic surface is exemplified by skin, surgical
sutures, mucosal membrane surface, or an epithelial surface. An inorganic
surface may be the surface of a pipe or pipeline, a floor, a table-top, a
counter-top, hospital equipment, or a wheel chair, etc. Non-limiting
examples of a pipe is an oil pipeline, a water pipeline, an ice machine
pipe, or a beverage dispensing pipe.

[0044] It is contemplated that the antimicrobial solutions of the present
invention will find particular usefulness as antimicrobial mouthwash
solutions. Such mouthwash solutions are contemplated to be useful both in
conjunction with dental procedures and oral sterilization as well as in
general dental and oral hygiene applications. Antimicrobial mouthwash is
becoming extremely important in the prevention of oral cavity infections
as well as aspiration pneumonia. Microbial organisms in the mouth
particularly around the teeth, embed themselves in biofilm and the
pathogenesis of infection and colonization is similar to that seen in,
for example, vascular catheters. In this regard, it is contemplated that
one will preferably apply the triple combinations of the present
invention, that will include an antimicrobial (possibly antiseptic) with
EDTA and low concentration alcohol as a mouthwash or mouth flush
solution.

[0045] The invention also provides a kit for disinfecting a surface to
reduce microorganisms thereon, wherein the kit comprises components
including at least one antimicrobial agent, at least one
anticoagulant/chelator, and at least one alcohol, contained in a suitable
container. The components may be combined in a single container, or
powdered components may be lyophilized, combined and separately
compartmentalized, or all of the components may be placed in separate
containers. In some embodiments, only the antimicrobial agent(s) is
included as a dried powder. In aspects comprising powdered components,
the kit may optionally include a second carrier solution for
reconstituting the lyophilized antibiotic agent(s).

[0046] In preferred aspects, the kit will include a unit dose of a
pharmacologically effective amount of minocycline and EDTA (or heparin),
either provided separately as a lyophilized or powdered dose or already
mixed in an ethanol solution. In a specific embodiment, the unit dose
contains at least about 9 mg of minocycline and at least about 90 mg of
EDTA. Such a kit may further comprise a preselected amount of an ethanol
solution such that when the ethanol solution is mixed with the
lyophilized unit dose, the concentration of minocycline is 3 mg/ml and
the concentration of EDTA is 30 mg/ml.

[0047] Kits in accordance with the present invention may be used to
reduce/eliminate microbes on the surface of a medical device, a pipe or
pipeline, a floor, a table-top, a counter-top, hospital equipment, or a
wheel chair. It is also contemplated that the kits of the invention will
further comprise a means for introducing the kit components into the
medical device, the pipe or surface.

[0048] In some specific aspects of the invention, a syringe or vial
comprising a lyophilized unit dose of a pharmacologically effective
amount of one or more of the three components of the flush solutions of
the present invention. For example, such a syringe may comprise
minocycline and EDTA (or heparin) mixed in an ethanol solution. In a
specific embodiment, the unit dose contains at least about 9 mg of
minocycline and at least about 90 mg of EDTA. Such a syringe or vial may
further comprises a preselected amount of an ethanol solution such that
when the ethanol solution is mixed with the lyophilized unit dose, the
desired concentration of the particular agent is obtained, such as about
3 mg/ml in the case of minocycline and about 30 mg/ml. in the case of
EDTA.

[0049] In other embodiments of the invention, a locking solution for
filling and/or flushing a medical indwelling device such as, but not
limited to, an implanted catheter is contemplated. The locking solution
may comprise at least one antimicrobial agent, at least one chelator
and/or anticoagulant, and at least one alcohol.

[0050] Some of the terms used in the present application are defined
below:

[0051] An "antimicrobial agent" is defined herein as an agent that has
antibiotic properties against bacteria, fungi, viruses and other
pathogens and includes antibacterial agents, antifungal agents, antiviral
agents and antiseptic agents.

[0052] As used herein, the term "antifungal agent" is defined as a
compound having either a fungicidal or fungistatic effect upon fungi
contacted by the compound. As used herein, the term "fungicidal" is
defined to mean having a destructive killing action upon fungi. As used
herein, the term "fungistatic" is defined to mean having an inhibiting
action upon the growth of fungi.

[0053] As used herein, the term "antibacterial agent" is defined as a
compound having either a bactericidal or bacteriostatic effect upon
bacteria contacted by the compound. As used herein, the term
"bactericidal" is defined to mean having a destructive killing action
upon bacteria. As used herein, the term "bacteriostatic" is defined to
mean having an inhibiting action upon the growth of bacteria.

[0054] As used herein, the term "antiviral agent" is defined as a compound
that can either kill viral agents or one that stops the replication of
viruses upon contact by the compound.

[0055] For the purposes of this disclosure, the phrase "effective amount"
or "therapeutically effective amount" is defined as a dosage sufficient
to induce a microbicidal or microbistatic effect upon the microbes
contacted by the composition on a surface.

[0056] The phrase "a chelator" denotes one or more chelators. As used
herein, the term "chelator" is defined as a molecule comprising nonmetal
atoms, two or more of which atoms are capable of linking or binding with
a metal ion to form a heterocyclic ring including the metal ion.

[0057] As used herein the terms "contact", "contacted", and "contacting",
or "exposed" and "exposure" are used to describe the process by which any
of the compositions disclosed in the present invention, comes in direct
juxtaposition with the surface of a medical device or any other surface
from which microbial growth is to be reduced or eradicated.

[0058] As used herein in the specification, "a" or "an" may mean one or
more. As used herein in the claim(s), when used in conjunction with the
word "comprising", the words "a" or "an" may mean one or more than one.
As used herein "another" may mean at least a second or more.

[0059] Other objects, features and advantages of the present invention
will become apparent from the following detailed description. It should
be understood, however, that the detailed description and the specific
examples, while indicating preferred embodiments of the invention, are
given by way of illustration only, since various changes and
modifications within the spirit and scope of the invention will become
apparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

[0060] The following drawings form part of the present specification and
are included to further demonstrate certain aspects of the present
invention. The invention may be better understood by reference to one or
more of these drawings in combination with the detailed description of
specific embodiments presented herein.

[0061] FIG. 1. Ethanol in combination with M-EDTA as a flush solution used
for 15 minutes or 24 hours, as indicated, tested against MRSA in biofilm.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0062] Microorganisms that attach themselves to inert surfaces, such as
medical devices including, vascular catheters, endotracheal tubes, Foley
catheters, biliary stents, nephrostomy tubes, prosthetic valves,
ventriculostomy or epidural catheters, or fluid pipelines, such as oil
pipelines or water pipelines, produce a layer made of exopolysaccharide
called microbial biofilm. These organisms embed themselves in this layer.
This biofilm layer ultimately becomes the protective environment that
shields these organisms on the inert surface from the antimicrobial
activity of various antibiotics or antiseptics. In U.S. Pat. Nos.
5,362,754 and 5,688,516, incorporated herein by reference in their
entirety, the present inventor demonstrated that a combination of one or
more antimicrobial agent with one or more chelator and/or anticoagulant
(such as EDTA or heparin) reduces or eradicates these
antibiotic-resistant biofilm embedded microorganisms if the antimicrobial
and chelator combination is allowed to dwell on the surface for at least
4 hours. However, in both clinical and environmental situations, it is
typically not feasible to allow a 4 hour dwell time for the chelator and
antimicrobial agent to reduce or eradicate the microbes. For example, it
is not possible to interrupt the therapy of critically ill patients
receiving continuous infusion therapy through a vascular catheter for 4
hours. It is also not possible to interrupt an environmental situation
involving fluid pipelines for 4 hours to allow for such a prolonged dwell
time of antimicrobial/chelator solution.

A. THE PRESENT INVENTION

[0063] The present invention allows rapid reduction and/or eradication of
microorganisms embedded in a biofilm in a time as short as about 15
minutes of exposure to combinations of at least one antimicrobial and at
least one chelator/anticoagulant, if this combination is prepared in an
alcohol. This is exemplified in one embodiment by minocycline-EDTA in an
ethanol solution, which is described in detail in the application.
However, one of skill in the art will recognize that one may use any
antimicrobial agent, any chelator/anticoagulant and any alcohol.

[0064] In addition, the present invention provides antimicrobial solutions
comprising one or more antimicrobial agents, one or more
chelator/anticoagulant, and an alcohol solution. The present invention
also provides methods for the rapid reduction or eradication of
microorganisms embedded in a biofilm on a surface comprising contacting
or exposing the surface to a flush solution of the invention. Thus, the
invention provides methods for reducing or eradicating microbes from the
surfaces of medical devices, including indwelling medial devices, as well
as other surfaces, pipelines and the like.

[0065] The compositions and the methods of the present invention have an
unexpected and surprising efficacy not provided by compositions that
comprise only alcohol solutions, or compositions that comprise
combinations of antimicrobials with chelators/anticoagulants. In one
specific example, the combination of the antimicrobial agent minocycline
with the chelator/anticoagulant EDTA requires about 4 hours of exposure
or dwell time to reduce microbes from the surface of a medical device. On
the other hand, a 25% ethanol solution alone suppresses colonizing
organisms embedded in biofilm, but does not eradicate them. However, one
exemplary composition of the present invention, comprising minocycline,
EDTA and 25% ethanol provides rapid reduction and/or eradication of the
microbial organisms within 15 minutes of exposure and also prevents the
re-growth of the microbes.

[0066] 1. Medical Applications

[0067] One of the applications of the antimicrobial flush solutions of the
invention is to reduce or eradicate microbes from the surfaces of medical
devices especially indwelling medical devices such as catheters,
endotracheal tubes, nephrostomy tubea, biliary stents, orthopedic
devices, prosthetic devices, and medical implants.

[0068] There are at least 5 million central venous catheters inserted
annually in the United States, 1.5 million of which are long-term
catheters that remain in place for an average of 100 days, and at least
3.5 million short-term catheters that remain for an average of 7 days.
All of these venous catheters are flushed with heparin on a daily basis.
It is estimated that at least 150-175 million catheter flushes occur
annually in the United States alone. Heparin has good anticoagulant
activity and, hence, prevents thrombotic occlusions. However, heparin has
no antimicrobial activity and, in fact, given the alkaline media that
heparin creates, it has been shown to be a promoter of microbial
colonization of catheter surfaces. Irrespective of whether heparin is
used, almost 90%-100% of indwelling vascular catheters end up being
colonized with organisms embedded in biofilm on the surface of these
devices, particularly at the lumenal surface. Hence, the most serious and
frequent complication of vascular catheters is infection, whereby as
fluid is flushed through the lumen of the catheter, microorganisms
migrate into the bloodstream and cause catheter-related bloodstream
infections. Indwelling central venous catheters are associated with
around 5%-8% frequency of catheter-related bloodstream infection, which
in turn is associated with an attributable mortality of 25% in critically
ill patients. Such an event is also associated with high morbidity and a
cost per episode of an average of $30,000.

[0069] EDTA is a well-known chelator of iron and calcium, as well as an
active anticoagulant used in blood collection tubes. EDTA has been shown
to have equivalent anticoagulant activity to heparin. In addition, EDTA
has antibiofilm activity and enhances the antimicrobial activity of other
antimicrobial agents, such as minocycline. However, for a combination of
an antmicrobial with a chelator (such as minocycline-EDTA) to eradicate
organisms embedded in biofilm, contacting the surface for at least 4-hour
with this combination is required. This is demonstrated in U.S. Pat. No.
5,362,754 (see especially data in Tables 3, 4 and 5) and in U.S. Pat. No.
5,688,516, (see especially columns 15 and 16 and Tables 3, 4 and 5). This
prolonged period of contacting or dwell time is not possible in the
highest risk patient population (i.e., in patients receiving total
parental nutrition or critically ill patients), as these patients require
a continuous, often uninterrupted, infusion through the catheter. In
order to allow for a rapid reduction or eradication of microorganisms, an
improvement has been developed in the present invention wherein the
antimicrobial(s) and chelator(s)/anticoagulant(s) is prepared in an
alcohol solution. In one example, this is embodied by a minocycline-EDTA
combination in a 25% ethanol solution.

[0070] The present invention, thus, provides that indwelling medical
devices such as catheters be flushed with this antimicrobial and
chelator/anticoagulant in an alcohol-based solution. This will provide
chelation/anticoagulation through the chelator (such as EDTA). In
addition, the combination of antibiotic/chelator with an alcohol results
in broad-spectrum reduction or eradication of microbial organisms
embedded in biofilm. The alcohol further increases the efficacy of the
combination.

[0071] Some examples of indwelling medical devices that may be treated
with the solutions of the present invention include abdominal cavity
drainage bags, connectors and tubing used by colostomy patients, vascular
shunts, orthopedic, intraocular, or penile prosthesis devices.
Angioplasty devices, heart valves and cardiac pacemakers also are
included within the present invention. Catheters such as urinary, venous,
arterial, and peritoneal catheters may be treated with the flush
solutions of the invention. In addition, tracheotomy devices, shunts,
surgical sutures, and other medical devices or prosthesis can be treated.

[0072] Furthermore, the medical devices which are amenable to coatings of
the compositions of the invention generally have surfaces composed of
thermoplastic or polymeric materials such as polyethylene, Dacron, nylon,
polyesters, polytetrafluoroethylene, polyurethane, latex, silicone
elastomers and the like. Devices with metallic surfaces are also amenable
to coatings with the antibiotic combinations. Such devices are
exemplified by bone and joint prosthesis. It is also contemplated that
the solutions of the invention will be used to disinfect organic surfaces
such as skin as well as mucosal surfaces.

[0073] An antimicrobial locking solution of the present invention may
comprise at least one alcohol, at least one antimicrobial agent and at
least one chelator and/or anticoagulant. Various antimicrobial substances
as disclosed herein and that are well known to one of ordinary skill in
the art may be combined with the locking solution in order to inhibit
infection. The antimicrobial locking solution of the present invention
may be use for filling or flushing a medical device such as an indwelling
device such as an implanted catheter. Other medical devices that are
contemplated for use in the present invention are disclosed herein.

[0074] 2. Environmental Applications

[0075] Other than reduction/eradication of microbes in medical devices,
the flush solutions of the present invention are also useful in the
eradication of the surfaces of other surfaces that microbes can grow on
such as pipes, pipelines etc. Fluid pipelines, such as oil and water
pipelines, are often obstructed by lumenal biofilm that is produced by
microorganisms that colonize the internal surface of these pipelines.
Often these pipelines are flushed with antimicrobial agents. However,
antimicrobial and antiseptic agents have little activity against
organisms embedded in biofilm. Tons of antibiotics, such as gentamicin,
are often used to flush the lumen of oil pipelines, to no avail. The
present invention provides new and effective compositions and methods for
the eradication of organisms, as well as biofilm embedding the lumen of
pipelines (oil, water), as well as other devices, such as ice machines.
These pipelines or machines can be flushed or rinsed with the
compositions of the invention that comprise at least one antimicrobial
agent and at least one chelator or anticoagulant prepared in a base
solution of ethanol. Flushing the pipelines, machines or tubes with the
compositions of the invention provide rapid reduction and/or eradication
of the biofilm and the organisms in biofilm thereby preventing any
obstruction or contamination of the water, oil or the ice machines in
certain environmental settings.

B. ANTIMICROBIAL AGENTS AND MICROBES

[0076] The present compositions are contemplated to have one or more
antimicrobial agents. "Antimicrobial agents" are defined herein as
antibacterial agents, antifungal agents, antiviral agents and/or
antiseptic agents.

[0077] While the invention is not limited to any particular antimicrobial
agent some exemplary classes and examples of antibacterial agents,
antifungal agents, antiviral agents as well as antiseptic agents are
described above in the section entitled "summary of invention." Of course
one of skill in the art will appreciate that any combination as well as
agents from the different types and classes of the antimicrobial agents
can be combined to prepare the solutions of the invention.

[0078] Some non-limiting exemplary bacterial and fungal microbes that can
be reduced or eradicated by the compositions and methods of the invention
include Staphyloccous species such as Staphylococcus epidermidis,
Staphylococcus aureus; Aspergllus species, such as Aspergillus flavus,
Aspergillus terreus; Fusarium oxysporum, Candida species, such as Candida
krusei, Candida parapsilosis, Candida tropicalis, Candida albicans and
Candida glabrata. In addition, viruses can also be eradicated.

C. CHELATORS AND/OR ANTICOAGULANTS

[0079] A chelate is the type of coordination compound in which a central
metal ion is attached by coordinate links to two or more nonmetal atoms
in the same molecule. Heterocyclic rings are thus formed during
chelation, with the metal atom as part of the ring. The molecule
comprising the nonmetal linking atoms is termed a chelator. Chelators are
used in various chemical applications, for example as titrating agents or
as metal ion scavengers. Chelators can be used to remove ions from
participation in biological reactions. For example, the well-known
chelator ethylenediamine-N,N,N',N',-tetraacetic acid (EDTA) acts as an
anticoagulant because it is capable of scavenging calcium ions from the
blood.

[0080] It has been previously shown that chelators have significant growth
inhibitory effect against several microbes. It is known that iron and
other trace metals are essential in the life cycle of microorganisms such
as fungi and bacteria. Without these trace metals, microbes are unable to
grow and reproduce. Although iron is abundant in nature, its availability
for microbial assimilation is limited owing to the insolubility of ferric
ions at neutral or alkaline pH. As a consequence, many microbes have
evolved their own specialized trace metal-scavenging molecules, called
siderophores, which bind with trace metals and make them available for
uptake by the microbes. The chelators used in conjunction with the
present invention provide an inhibitory effect upon microbial pathogens
by competing with the siderophores for any available trace metal ions. In
this way, the chelators present in the pharmaceutical preparations of the
present invention "steal" the metal ions essential for microbial growth,
effectively causing the microbe to "starve to death." The additional
antibiotic agents and the ethanol of the compositions of the present
invention then come in and attack the weakened microbe, thereby
destroying them or inhibiting their growth.

[0081] Table 1 below provides a representative list of chelators useful in
conjunction with the present invention. However, the list provided in
Table 1 is not meant to be exhaustive. Preferred chelators are those
which bind trace metal ions with a binding constant ranging from 101
to 10100. More preferred chelators are those which bind trace metal
ions with a binding constant ranging from 1010 to 1080; and
most preferred chelators are those which bind trace metal ions with a
binding constant ranging from 1015 to 1060. Furthermore,
preferred chelators are those which have been shown to have an inhibitory
effect upon target microbial pathogens, for example the disodium salt of
EDTA.

[0083] The flush solutions of the instant invention are contemplated to
comprise an alcohol, such as an antiseptic or disinfectant alcohol.
Exemplary alcohols include ethanol, methanol, isopropanol, benzyl
alcohol, chlorobutanol, phenylethyl alcohol,
2-bromo-2-nitropropan-1,3-diol, and the like. The present invention
contemplates any effective concentration of alcohol, but will typically
employ a final alcohol concentration in the range of 5%-80% (v/v), more
preferably in the range of 10% to 50%, more preferably in the range of
15% to 40%, more preferably in the range of 20% to 30%, with the most
preferable being about 25%. Thus, the more preferred concentration of
alcohol will include 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%,
40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80% (v/v) of the alcohol in
the preparation of the instant antimicrobial solutions. This includes the
use of intermediate concentrations of alcohol such as 11%, 22.5%, 26% and
the like.

[0084] Alcohols such as ethanol are long known to have disinfectant
properties. In EP1245247 and U.S. Pat. No. 6,350,251, it is reported that
the combination of ethanol with EDTA provides a biocidal lock for
indwelling medical devices. In contrast, it has also been shown that a
combination of ethanol with EDTA is less effective in killing microbes
than ethanol alone (Sherertz et al., 2002). Thus, the art is in a flux
about the exact role of the combination of ethanol with EDTA.

[0085] The present inventor has shown that ethanol alone, while requiring
only a relatively short duration of contact, is only partially effective
in killing or controlling microbes on the surface of an indwelling
medical device or other surface. In contrast, a combination of an
antimicrobial agent and a chelator such as EDTA may be effective, yet it
requires a somewhat longer duration of contact (e.g., sometimes on the
order of 4 hours). However, in the present invention it is shown that the
triple combination of an alcohol, an antimicrobial and a
chelator/anticoagulant provides unexpectedly effective anti-microbial
properties in a very short duration and in addition to eradicating
microbes rapidly from a surface they also preventing re-growth of the
microbial pathogen on the surface. An additional advantage for the triple
combination, as shown in the studies set forth herein below, is that it
is effective at eradicating a broader range of microbial organisms
(bacteria and fungi), even at the shorter durations of contact with the
treated surface.

E. ADDITIONAL AGENTS

[0086] It is also contemplated that any additional pharmacologically
active ingredients or sterilization agents may be comprised in the
solutions of the present invention or may be used separately for flushing
or treating the devices of the present invention to further reduce or
eliminate pathogenic microbes and viruses. Typical pharmacologically
active ingredients include antifibrin agents, anti-thrombotic agents, and
anti-inflammatory agents. Anti-inflammatory agents include steroids, and
nonsteroidal anti-inflammatory agents, and salicylates. Anti-thrombotic
drugs including acetylsalicylic acid, dipyridamole, heparin, ibuprofen,
indomethacin, prostaglandins, sulfinpyrazone, warfarin, thrombolytic
enzymes such as streptokinase, urokinase, or plasminogen activator.
Complexing agents such as ammonium-1-pyrrolidine dithiocarbanate may also
be used. However, the above examples are not meant to be limiting.

[0087] In certain applications, it will be sufficient to provide a single
pharmacologically active ingredient in the device. In other situations,
it will be desirable to combine compatible ingredients. For example, it
may prove useful to provide an antimicrobial agent along with an
anticoagulant and/or an anti-inflammatory agent. In another example, it
may prove useful to provide multiple antimicrobial agents with differing
target specificities, modes of action or duration, either alone or in
combination with anticoagulants or antiinflammatory agents.

F. PACKAGING AND KITS

[0088] Described herein are various packaging techniques that may be
employed in providing the flush solutions of the invention as part of a
commercially available kit. The kit will optionally include an
instruction sheet insert to identify how the kit is to be used.

[0089] The kits described in this section are exemplified by a solution
comprising minocycline as the antibiotic, EDTA as the
chelator/anticoagulant, and ethanol. However, as will be appreciated by
the skilled artisan, any other combination of one or more antibiotic, one
or more chelator/anticoagulant, and ethanol as described in the present
disclosure may be packaged in a similar manner. The kit may comprise of
one or two or three or more compartments. The components of the kit may
be provided in separate compartments or in the same compartment. The
components of the kit may be provided separately or mixed. The mixed
components may contain two or more agents such as an antibiotic, a
chelator/anticoagulant, or ethanol, or additional component.

[0090] One of the packaging options below maintain the ingredients, for
example, the antibiotic, such as minocycline, and the chelating
agent/anticoagulant, such as EDTA, in an uncombined form. These
components are to be combined shortly before use. These packaging options
are contemplated to be part of a 2-compartment or three-compartment
container system to provide a total volume of about 3 ml of the ready to
use preparation. Any compartmentalized container system may be used to
package the compositions of the present invention. An exemplary container
system is available from Becton Dickinson.

[0091] Option 1: A 3-Compartment system comprising two dry components such
as 3-9 mg minocycline (dry), 10-100 mg EDTA (powdered) and one wet
component comprising 3 ml diluent (alcohol alone or diluted in saline or
distilled water). When ready for use, the dry components, minocycline and
EDTA, will be allowed to mix with the diluent. Final concentration of the
mixture should be about 3 mg/ml minocycline and 30 mg/ml EDTA.

[0092] Option 2: A 2-Compartment system antibiotic and
chelator/anticoagulant (one wet, one dry) comprising for example 3-9
mg/ml minocycline and 10-100 mg EDTA. When ready for use, the dry EDTA
powder will be combined with the minocycline in solution. The minocycline
may be suspended in either saline, distilled water, alcohol solution or
other physiologically acceptable diluent. Alternatively, the minocycline
may be in a dry powdered form, and the EDTA in solution. A wet/wet®
dual chamber container system, available from Becton-Dickinson, may be
used in these applications.

[0093] Option 3: A 2 compartment system comprising both wet compartments
comprising antimicrobial agent(s) and chelator/anticoagulant comprising
in one example 10-100 EDTA Solution and 3-9 mg/ml Minocycline Solution
where the solution comprises alcohol. When ready for use, the EDTA
solution will be combined with minocycline solution. Once combined, the
solution will have a concentration of 3 mg/ml minocycline and 30 mg/ml
EDTA. A wet/wet® dual chamber container system, available from
Becton-Dickinson, may be used in these applications.

[0094] Option 4: A 2 compartment system both comprising dry powders of the
antimicrobial agent(s) and chelator/anticoagulant in a diluent comprising
for example, 10-100 EDTA (dry) and 3-9 mg minocycline (dry) and diluent
solution. The dry EDTA and dry minocycline may be suspended in a solution
of an alcohol made in either saline, distilled water, or other
physiologically acceptable diluent. A liquid/dry® dual container
system, from Becton-Dickinson, may be used. When ready for use, the dry
minocycline powder will be allowed to combine with the EDTA solution. The
EDTA can be suspended in either saline or distilled water, or alcohol
solution, or other physiologically acceptable diluent.

[0095] The various compartmentalized embodiments of the present invention
as disclosed above, may be provided in a kit form. Such kits would
include a container means comprising a volume of diluent, comprising an
alcohol optionally diluted if required in a solution such as saline or
sterile water, a second (or more) container means comprising one or more
antimicrobial or biocide, a third (or more) container means comprising
one or more chelating/anticoagulant agent. The dry components may
optionally be mixed in one compartment. The addition of the diluent would
then be performed immediately prior to use.

[0096] The container means of the kits will generally include at least one
vial, test tube, flask, bottle, syringe or other container means, into
which the antimicrobial/chelator/anticoagulant/alcohol may be placed, and
preferably, suitably aliquoted. Where a second or third antibiotic agent,
other chelator, alcohol, or additional component is provided, the kit
will also generally contain a second, third or other additional container
into which this component may be placed. The kits of the present
invention will also typically include a means for containing the alcohol,
antimicrobial agent, chelator/anticoagulant, and any other reagent
containers in close confinement for commercial sale. Such containers may
include injection or blow-molded plastic, or glass containers into which
the desired vials are retained.

G. EXAMPLES

[0097] The following examples are included to demonstrate preferred
embodiments of the invention. It should be appreciated by those of skill
in the art that the techniques disclosed in the examples which follow
represent techniques discovered by the inventor to function well in the
practice of the invention, and thus can be considered to constitute
preferred modes for its practice. However, those of skill in the art
should, in light of the present disclosure, appreciate that many changes
can be made in the specific embodiments which are disclosed and still
obtain a like or similar result without departing from the spirit and
scope of the invention.

[0098] In vitro Model of Colonization (Modified Robbins Device). The in
vitro model utilized a modified Robbins device (MRD) to study the
colonization of catheter segments with organisms embedded in biofilm. The
modified Robbins device has been previously described (Nickel et al.,
1991; Evans et al., 1987, see also U.S. Pat. No. 5,362,754) and is
constructed from an acrylic block, 42 cm long with a lumen of 2×10
mm. It consists of 25 evenly spaced specimen plugs, each connected to a
silicone catheter segment (Allegiance Healthcare Corp., McGaw Park, Ill.)
whose anterior surface (0.3 cm2) comes in conta-ct with the flushed
infusate. After placing the catheter segments in the specimen plug of the
modified Robbins device, the entire apparatus was gas sterilized using
ethylene oxide. A 500 ml 5% dextrose in water (5% D5/W) was
connected to the modified Robbins device through an intravenous tubing
administration set and was subsequently infected with an innoculum of
108 CFU/ml of methicillin-resistant Staphylococcus aureus (MRSA), to
produce an infected infusate at the concentration of 2×105
CFU/ml. The biofilm-producing S. aureus isolates were obtained from
patients with CRBSI. In another series of experiments, 500 ml 5%
D5/W bag was infected with a biofilm-producing C. parapsilosis using
an inoculum of 105 CFU/ml to produce an infected infusate at a
concentration of 2×102 CFU/ml of C. parapsilosis. The whole
system was incubated at 37° C. and the infected infusate was
flushed through the MRD using a peristaltic pump permitting the infusate
to flow at the rate of 60 ml/hour for 8 hours. The modified Robbins
device was left to incubate for a total of 18 hours (another 10 hours).
Subsequently, the infected bag was removed and a 250 ml saline sterile
bag which was infused through the MRD at 125 ml/hour for 2 hours in order
to remove all free floating organisms. To insure biofilm formation, at
least three catheter segments were randomly removed from the 25 evenly
spaced catheter segments in the MRD and studied by scanning electron
microscopy. This was repeated for every organism tested.

[0099] Exposure to Anticoagulants/Antimicrobials. The remaining catheter
segments were removed and each segment was placed in a tube containing 2
ml of one of the following broth solutions: (1) Mueller-Hinton broth
(Becton Dickinson & Co., Cockeysville, Md.); (2) EDTA at a concentration
of 30 mg/ml in broth (Abbott Laboratories, North Chicago, Ill.); (3)
minocycline at 3 mg/ml in broth (Wyeth-Ayerst Laboratories, Collegeville,
Pa.); (4) minocycline (3 mg/ml) and EDTA combination (M-EDTA) in broth;
(5) 25% ethanol (ETOH) solution in broth; (6) minocycline at 3 mg/ml in
25% ethanol solution in broth; (7) EDTA 30 mg/ml in 25% ethanol solution
in broth; and (8) M-EDTA in 25% ethanol solution in broth. The
experiments were repeated in triplicate or quadruplicate, and during each
experiment, 2-5 catheter segments were exposed to the same solution for
only 15 minutes at 37° C. Subsequently, some of the catheter
segments were immediately removed and cultured by scrape sonication.
Other alternating catheter segments were removed, placed in broth (TSB),
incubated for 24 hours, and then cultured by scrape sonication. This
added step of re-incubating catheter segments in broth after the 15
minutes exposure was done to determine whether these agents suppressed
the growth of organisms embedded in biofilm or eradicated them. The
surface of the catheter segment that was exposed to the infected infusate
was scraped with a sterile wooden applicator stick and placed, along with
the stick, in a tube containing 0.5 ml of trypticase soy broth. The tubes
were sonicated for five minutes; 0.1 ml of the sonicated broth solution
in the tube was pipetted and plated over a blood agar plate, which was
incubated at 37° for 24 hours. The agar plates were checked for
any contaminants. The isolated organisms had to be of the same species
and colonial morphology as the original organism used to infect the
infusate. The number of colonies quantitated from the agar plate was
multiplied by five to correct for the dilution factor and to determine
the total number of colonies isolated from a particular catheter segment.
A confluent growth of 100 or greater was calculated as ≧500
colonies.

[0100] Definitions. Inhibitory activity or suppression is defined as no
growth of microbial organisms immediately after 15 minute exposure to the
antimicrobial solution. However, regrowth of the organisms after 24 hours
incubation in broth was observed.

[0101] Eradication is defined as no growth of organisms after immediate 15
minutes exposure to the antimicrobial solution with no subsequent growth
upon reincubation for 24 hours in broth.

Results

[0102] As shown in Table 2, EDTA alone failed to eradicate
methicillin-resistant S. aureus and C. parapsilosis organisms embedded in
biofilm after 15 minutes of exposure, resulting in re-growth after 24
hours of incubation of the catheter segments in broth solution.
Minocycline alone (at a concentration of 3 mg/ml) with or without EDTA
resulted in some decrease in colonization. However, organisms continued
to grow after 15 minutes of exposure and after 24 hours re-incubation in
broth at 37° C. A 25% ethanol solution suppressed growth initially
to a mean concentration level of 138 colony forming units (CFU). However,
upon re-incubation in broth at 37° C. for 24 hours, there was
complete re-multiplication and growth of the staphylococcal organisms
embedded in biofilm to a high level of 500 CFU per catheter segment,
which is comparable to the growth of control catheter segments. The
combination of EDTA and 25% ethanol solution resulted in a significant
decrease in colonization immediately after 15 minutes of exposure to this
solution. However, regrowth occurred after re-incubation in broth
solution at 37° C. for an additional 24 hours. Minocycline in 25%
ethanol, with or without EDTA, resulted in complete eradication of
microorganisms embedded in biofilm after 15 minutes of exposure to the
solutions. In addition, re-incubation of the catheter segments in broth
for an additional 24 hours at 37° C. failed to allow regrowth of
the organisms, verifying the complete eradication of the S. aureus
organisms embedded in biofilm.

[0103] As shown in Table 3, EDTA alone, minocycline alone and M-EDTA
failed to eradicate C. parapsilosis organisms embedded in biofilm. A 25%
ethanol solution with or without minocycline, inhibited C. parapsilosis
growth after 15 minutes of exposure. However, regrowth was noted after 24
hours incubation in broth. EDTA in 25% ethanol and M-EDTA in 25% ethanol
completely eradicated C. parapsilosis in biofilm after 15 minutes
exposure with no regrowth after reincubation in broth.

[0104] Minocycline alone, EDTA alone or the combination of minocycline and
EDTA failed to eradicate organisms embedded in biofilm after a rapid
exposure of only 15 minutes. 25% ethanol solution also failed to
eradicate organisms embedded in biofilm and a high level of regrowth was
apparent after catheter segments were re-incubated in broth for an
additional 24 hours at 37° C.

[0105] The combination of ethanol/EDTA did achieve inhibition or
suppression of organisms embedded in biofilm after 15 minutes of exposure
of the catheter surfaces to this solution. However, regrowth was noted
upon re-incubation of the catheter segments in broth for 24 hours at
37° C. The combination of EDTA/25% ethanol, however, was superior
in its inhibitory activity when compared to 25% ethanol alone.

[0106] The combination of minocycline in 25% ethanol with or without EDTA
was highly active in eradicating organisms embedded in biofilm after 15
minutes of exposure to this combination. Regrowth of C. parapisilosis
occurred occasionally after exposure to minocycline in 25% ethanol.
Regrowth failed to occur after exposure to M-EDTA in 25% ethanol,
verifying the complete eradication of S. aureus organisms embedded in
biofilm after rapid exposure to this triple combination.

[0107] Because EDTA has anticoagulant activity and, in these experiments,
seems to have added to the antimicrobial activity of 25% ethanol, it was
prudent to use the triple combination of minocycline/EDTA in 25% ethanol
as a flush or antibiotic lock solution of central venous catheters. In
contrast, vancomycin alone or in combination with heparin failed to
eradicate microbial organisms embedded in biofilm from catheter surfaces,
even after a dwell time of 4-24 hours (see U.S. Pat. No. 5,362,574,
columns 11 and 12, Tables 3, 4 and 5.)

[0108] The inventor next determined the efficacy of minocycline and EDTA
combination in 25% ethanol in eradicating staphylococci and candida
embedded in biofilm. Prevention of regrowth after reincubation was
assessed using a novel silicone disk bioprosthetic colonization model.
The procedure is described below.

Experimental Procedure

[0109] On day 1, pieces of biofilm were prepared. Sterile (Ethylene Oxide
Gas Sterilized) silicone disks were placed in 5 ml sterile snap top
Falcon tubes and 0.5 ml of pooled plasma added. This was followed by
incubation (while rocking) overnight at 37° C.

[0110] On day 2, the bacteria was added to form the biofilm. Using sterile
plastic transfer pipettes, the plasma was suctioned out from the tubes
and replaced with 0.5 ml of bacterial inoculum (50 ml of Mueller-Hinton
broth containing 4-5 colonies of freshly grown bacteria). The tubes were
incubated overnight at 37° C.

[0111] On day 3, a drug was added in an attempt to kill the bacteria.
Before adding the drug, the pieces were washed in 0.5 ml of 0.9% saline
in order to remove any planktonic bacteria. The tubes (containing the
biofilm disks and saline) were placed in the incubator at 37° C.
for 30 minutes. The saline was then pipetted out using sterile plastic
transfer pipettes (taking care not to disturb the pieces too much). The
silicone disks were then transferred to new 5 ml snap-top falcon tubes
containing 0.5 ml of the drug solution to be tested. The drug solutions
tested were as follows: (1) minocycline 3 mg/ml; (2) EDTA 30 mg/ml; (3)
25% ethanol solution; (4) EDTA 30 mg/ml in 25% ethanol; (5) minocycline 3
mg/ml in 25% ethanol; (6) minocycline 3 mg/ml with EDTA 30 mg/ml; and (7)
triple combination of minocycline 3 mg/ml and EDTA 30 mg/ml in 25%
ethanol solution. The disks were allowed to sit in the drug for 1 hour.
The drug was then suctioned out using a plastic transfer pipette. The
pieces were once again washed with 0.5 ml saline (added, and shaken for
30 seconds). The disks were then transferred to 15 ml snap-top falcon
tubes containing 5 ml of 0.9% saline. The pieces were sonicated for 5
minutes, and then vortexed for 30 seconds. 100 microliters (u1) of the
saline was then plated on a room temperature TSAII blood agar plate, and
evenly spread using a sterile glass spreader. The plates were incubated
overnight at 37° C.

[0112] For the 24 hour reincubation studies, pieces of biofilm were
prepared the same exact way as the regular pieces. Before adding the
drug, the pieces were washed in 0.5 ml of 0.9% saline in order to remove
any planktonic bacteria. The tubes (now containing the biofilm disks and
saline) were placed in the incubator at 37° C. for 30 minutes. The
saline was then pipetted out using sterile plastic transfer pipettes
(taking care not to disturb the pieces too much). The silicone disks were
then transferred to new 5 ml snap-top falcon tubes containing 0.5 ml of
the drug to be tested. The disks were allowed to sit in the drug for 15
minutes. The drug was then suctioned out using a plastic transfer
pipette. The pieces were once again washed with 0.5 ml saline (added, and
shaken for 30 seconds). The pieces were then transferred to new sterile 5
ml snap-top falcon tubes containing 0.5 ml of sterile trypticase soy
broth (TSB) and then placed in the incubator at 37° C. overnight.

[0113] On day 4, colonies were counted and the results recorded. The
colonies were hand counted, and counting was stopped at 100 colonies.
Anything greater was considered >100 colonies. The counts were
recorded, and multiplied by a factor of 50 because of the dilution factor
between the 5 ml of saline containing the disk and the 100 μl that was
plated onto the TSAII blood agar plates. The pieces were then sonicated
(in the same TSB that grew overnight) for 5 minutes. 100 μl was then
plated on TSAII blood agar plates, and the plates were placed in the
incubator to grow overnight at 37° C.

[0114] On day 5, the regrowth pieces were counted. The colonies were hand
counted, and counting was stopped at 100 colonies. Anything greater was
considered >100 colonies. The counts were recorded, and multiplied by
a factor of 5 because of the dilution factor between the 0.5 ml of TSB
containing the disk and the 100 μl that was plated onto the TSAII
blood agar plates.

Results

[0115] The silicone disk bioprosthetic colonization model has been
previously described by Kuhn et al. (2002). This in vitro model is more
clinically relevant than the modified Robin device, in vitro model, in
that it allows the silicone disk segments to be immersed in serum prior
to exposing to high inoculum of bacteria or fungi. Furthermore, it allows
a higher concentration of adherence of bacteria and fungi on the silicone
disk of up to 5,000 CFU/disk (the modified Robbins device allows for only
500 CFU/latex catheter segment). Because of the high inoculum that the
silicone disk segments were exposed to in the bioprosthetic colonization
model, the various disk segments were exposed to the various
antimicrobial agents for one hour (rather than 15 minutes in the modified
Robbins device). The results were consistent with the findings and
observations in the modified Robbins device model. As shown in Table 4,
exposure to either minocycline alone or EDTA or ethanol, or the
combination of minocycline and EDTA, failed to suppress the bioprosthetic
MRSA colonization of the silicone disks. EDTA in 25% ethanol had some
partial suppression but there was regrowth of the organisms after 24 hour
incubation. As expected, the control silicone disk segments were heavily
colonized before and after 24 hour reincubation. Minocycline in 25%
ethanol was highly suppressive but there was regrowth after 24 hour
incubation. However, the triple combination of M-EDTA in 25% ethanol was
unique in completely eradicating the MRSA organisms, with complete
inhibition of regrowth after 24 hours of incubation.

[0116] Table 5 shows a similar trend for Candida parapsilosis. Minocycline
alone, EDTA alone, or the combination of M-EDTA failed to suppress or
eradicate the growth of Candida parapsilosis on silicone disks.
Furthermore, there was a heavy regrowth of the C. parapsilosis on
silicone disks after exposure to these agents and reincubation for 24
hours. Twenty-five percent ethanol alone, EDTA in 25% ethanol or
minocycline in 25% ethanol failed to completely suppress Candida
parapsilosis growth after one hour exposure and there was heavy regrowth
after 24 hour reincubation. The triple combination of M-EDTA in 25%
ethanol completely eradicated the organisms on the silicone disks after 1
hour exposure. Furthermore, the level of regrowth associated with a
triple combination after 24 hours of reincubation was significantly lower
than all the other alternative agents or their dual combination,

[0117] Thus, the two in vitro models of colonization (the modified Robbins
device as well as the silicone disk bioprosthetic colonization model)
show that the triple combination is uniquely and highly effective in
eradicating organisms embedded in biofilm on latex and silicone polymers
with minimal or no regrowth after 24 hour exposure to the combination.
These two models are predictive of the clinical efficacy of this triple
combination in eradicating organisms embedded in biofilm on catheters at
a temperature of 37° C.

[0118] Hence, the triple combination is superior in efficacy to the
combination of minocycline and EDTA, EDTA and ethanol or minocycline and
ethanol.

[0119] All of the compositions and/or methods and/or apparati disclosed
and claimed herein can be made and executed without undue experimentation
in light of the present disclosure. While the compositions and methods of
this invention have been described in terms of preferred embodiments, it
will be apparent to those of skill in the art that variations may be
applied to the compositions and/or methods and/or apparati and in the
steps or in the sequence of steps of the method described herein without
departing from the concept, spirit and scope of the invention. More
specifically, it will be apparent that certain agents which are both
chemically and physiologically related may be substituted for the agents
described herein while the same or similar results would be achieved. All
such similar substitutes and modifications apparent to those skilled in
the art are deemed to be within the spirit, scope and concept of the
invention as defined by the appended claims.

REFERENCES

[0120] The following references, to the extent that they provide exemplary
procedural or other details supplementary to those set forth herein, are
specifically incorporated herein by reference.

[0121] U.S. Provisional
Patent Application Ser. No. 60/261,447

[0122] U.S. Provisional Patent
Application Ser. No. 60/316,165

[0123] U.S. Non-Provisional patent
application Ser. No. 10/044,842

[0124] U.S. Pat. No. 5,362,754

[0125]
U.S. Pat. No. 5,688,516

[0126] U.S. Pat. No. 6,350,251

[0127] Bleyer et
al., In: Proceedings of the 4th Decennial International Conference on
Nosocomial and Healthcare-Associated Infections in conjunction with the
10th Annual Meeting of the Society for Healthcare Epidemiology of
America, Atlanta, Ga., pp 91, 2000.